The present invention comprises small molecular weight, non-peptidic inhibitors of Protein Tyrosine Phosphatase 1 (PTP1) which are useful for the treatment and/or prevention of Non-Insulin Dependent Diabetes Mellitus (NIDDM).
The mechanism of insulin action depends critically upon the phosphorylation of tyrosine residues in several proteins in the insulin signalling cascade. Enzymes that dephosphorylate these proteins, protein tyrosine phosphatases (PTPs), are important negative regulators of insulin action. Therefore, the use of specific PTP inhibitors may therapeutically enhance insulin action.
The insulin resistance that is central to noninsulin-dependent diabetes mellitus (NIDDM) appears to involve a defect in an early process in insulin signal transduction rather than a structural defect in the insulin receptor itself. (J. M. Olefsky, W. T. Garvey, R. R. Henry, D. Brillon, S. Matthai and G. R. Freidenberg, G. R. (1988).) Cellular mechanisms of insulin resistance in non-insulin-dependent (Type II) diabetes. (Am. J. Med. 85: Suppl. 5A, 86-105.) A drug that improved insulin sensitivity would have several advantages over traditional therapy of NIDDM using sulfonylureas, which do not alleviate insulin resistance but instead compensate by increasing insulin secretion.
The binding of insulin to the xcex1-subunits of the insulin receptor permits the xcex2-subunits to catalyze phosphorylation of target proteins on tyrosine residues. There are 22 tyrosine residues in each insulin receptor xcex2-subunit itself and autophosphorylation of at least 6 of these tyrosines, in 3 distinct domains, is known to be involved in insulin action. (C. R. Kahn (1994) Insulin action, diabetogenes, and the cause of type II diabetes. Diabetes 43: 1066-1084.) Autophosphorylation of Tyr960 in the juxtamembrane domain is important for receptor internalization and for the interaction of the receptor with downstream signalling molecules such as insulin receptor substrate 1 (IRS-1).) (T. J. O""Neill, A. Craparo and T. A. Gustafson (1994) Characterization of an interaction between insulin receptor substrate 1 and the insulin receptor by using the two-hybrid system. Mol. Cell Biol. 14: 6433-6442.) Autophosphorylation of tyrosine residues 1146, 1150 and 1151 in the regulatory domain permits continued tyrosine kinase activity of xcex2-subunits, even after insulin has dissociated from the xcex1-subunits, and activates the kinase toward other protein substrates. (R. Herrera and O. M. Rosen (1986) Autophosphorylation of the insulin receptor in vitro: designation of phosphorylation sites and correlation with receptor kinase activation. J. Biol. Chem. 261: 11980-11985.) Deletion of autophosphorylation sites at Tyr1316 and Tyr1322 in the C-terminal domain attenuates the metabolic actions of insulin, but augments its mitogenic actions. (H. Maegawa, D. McClain, G. Freidenberg, J. Olefsky, M. Napier, T. Lipari, T. Dull, J. Lee, and A. Ullrich (1988) Properties of a human insulin receptor with a COOH-terminal truncation. II. Truncated receptors have normal kinase activity but are defective in signalling metabolic effects. J. Biol. Chem. 263: 8912-8917.) (Y. Takata, N. J. G. Webster, and J. M. Olefsky (1991) Mutation of the two carboxyl-terminal tyrosines results in an insulin receptor with normal metabolic signalling but enhanced mitogenic signalling properties. J. Biol. Chem. 266: 9135-9139.) Dephosphorylation of these autophosphorylated sites occurs rapidly in vivo, suggesting that a protein tyrosine phosphatase (PTPase) is involved in terminating insulin action. A compound that inhibited this PTPase, therefore, should potentiate insulin action. Indeed, vanadate potentiates insulin action, at least in part, by such a mechanism (Y. Schechter (1990). Insulin-mimetic effects of vanadate. Possible implications for future treatment of diabetes. Diabetes 39: 1-5.) The PTPase(s) that act on the insulin receptor, however, has not been identified definitively.
It has been estimated that the human genome encodes as many as 500 PTP enzymes (T. Hunter (1995) Protein kinases and phosphatases: The Yin and Yang of protein phosphorylation and signalling. Cell 80:225-236), but less than 100 have been identified and have been grouped into 4 sub-families (E. A. Fauman and M. A. Saper (1996) Structure and function of the protein tyrosine phosphatases. Trends Biochem. Sci. 21:413-417.) Members of the tyrosine-specific PTP sub-family are further divided into the receptor PTPases (such as CD45 and LAR) which typically have a large variable extracellular domain, a single transmembrane spanning region, and two intracellular phosphatase catalytic domains and the non-receptor PTPases. This latter group includes PTP that resemble PTP1. (D. A. Pot and J. E. Dixon (1992) A thousand and two protein tyrosine phosphatases. Biochim. Biophys. Acta 1136: 35-43.) There is data to support the proposition that the insulin receptor PTPase may be PTP1-like. For instance, an insulin-dependent association of PTP1 with insulin receptors has been described. (D. Bandyopadhyay, A. Kursari, K. A. Kenner, F. Liu, J.Chernoff, T. A. Gustafson, J. Kusari (1997) Protein-tyrosine phosphatase 1B complexes with the insulin receptor in vivo and is tyrosine-phosphorylated in the presence of insulin. J. Biol. Chem. 272: 1639-1645; and L. Seely, et al. (1996) Protein tyrosine phosphatase 1B interacts with the activated insulin receptor. Diabetes 45:1379.) Furthermore, PTP1 dephosphorylates purified insulin receptors sequentially in the order observed in vivo (i.e., Tyr1150=Tyr1151 greater than Tyr1146), (C. Ramachandran, R. Aebersold, N. Tonks and D. A. Pot (1992) Sequential dephosphorylation of a multiply phosphorylated insulin receptor peptide by protein tyrosine phosphtases. Biochemistry 31: 4232-4238) and insulin acutely increases PTP1 mRNA in hepatoma cells. (N. Hashimoto and B. J. Goldstein (1992) Differential regulation of mRNAs encoding three protein-tyrosine phosphatases by insulin and activation of protein kinase C. Biochem. Biophys. Res. Commun. 188: 1305-1311.) Insulin resistance induced in Rat 1 fibroblasts by high glucose (27 mM) is preceded by an approximate doubling of cytosolic PTP1 activity that is blocked by the insulin-sensitizer, pioglitazone. (H. Maegawa, R. Ide, M. Hasegawa, S. Ugi, K. Egawa, M. Iwanishi, R. Kikkawa, Y. Shigeta, and A. Kashiwagi (1995) Thiazolidinedione derivatives ameliorate high glucose-induced insulin resistance via the normalization of protein tyrosine phosphatase activities. J. Biol. Chem. 270: 7724-7730.) Thus, a specific inhibitor of PTP1 could be used to potentiate insulin action. While there are no known small molecules that specifically inhibit PTP1, it was found that osmotic loading of hepatoma cells with neutralizing antibodies against PTP1b (the human homologue of rat PTP1) resulted in increased autophosphorylation of insulin receptors and phosphorylation of IRS-1 in response to insulin. (F. Ahmad, P.-M. Li, J. Meyerovitch, and B. J. Goldstein (1995) Osmotic loading of neutralizing antibodies demonstrates a role for PTPase 1B in negative regulation of the insulin signalling pathway. Diabetes 44: Suppl. 1 104A.) See also B. J. Goldstein (1993) Regulation of insulin receptor signaling by protein-tyrosine dephosphorylation. Receptor 3: 1-15.)
International Publication No. WO 96/30332, xe2x80x9cO-Malonyltyrosyl Compounds, O-Malonyltyrosyl Compound-Containing Peptides, and Uses thereof,xe2x80x9d published Oct. 3, 1996, disclose non-phosphorus containing O-malonyltyrosyl compounds, derivatives thereof, uses of the O-malonyltyrosyl compounds in the synthesis of peptides, and O-malonyltyrosyl compound-containing peptides. The O-malonyltyrosyl compounds and O-malonyltyrosyl compound-containing peptides are disclosed as being useful as inhibitors of protein-tyrosine phosphatase; however, no specific non-peptidic compounds or data is disclosed.
International Publication No. WO 96/23813, xe2x80x9cPeptides and Compounds that Bind to SH2 Domains,xe2x80x9d published Aug. 8, 1996, discloses tyrosine-containing peptides and compounds which bind to the SH2 domain or domains of various proteins, as well as methods for identifying such peptides and compounds. These peptides and compounds have application as agonists and antagonists of SH2 domain containing proteins, and as diagnostic or therapeutic agents for the diagnosis or treatment of disease conditions.
International Publication No. WO 96/40113, xe2x80x9cPhosphatase Inhibitors,xe2x80x9d published Dec. 19, 1996, discloses heterocyclic nitrogen containing compounds, such as nitropyridine or nitrothiazole, capable of inhibiting protein tyrosine phosphatase activity. Such molecules are disclosed as being useful to modulate or regulate signal transduction by inhibiting protein tyrosine phosphatase activity and to treat various disease states including diabetes mellitus.
International Publication No. WO 96/40109, xe2x80x9cMethods of Inhibiting Phosphatase Activity and Treatment of Disorders Associated Therewith Using Napthopyrones and Derivatives Thereof,xe2x80x9d published Dec. 19, 1996, discloses the use of naphthopyrone compounds to inhibit protein tyrosine phosphatase activity. Such compounds are disclosed as being useful to modulate or regulate signal transduction by inhibiting protein tyrosine phosphatase activity and to treat various disease states including diabetes mellitus.
The compounds of the present invention have surprising activity in that they are small molecular weight and non-peptidic compounds.
A compound of formula I or II 
wherein G1 is
a) xe2x80x94R2, or
b) xe2x80x94NR8R4;
wherein G2 is
a) CONHR3,
b) H,
c) CH2OH, or
d) CHxe2x95x90CHR3;
wherein R1 is
a) xe2x80x94OSO3H,
b) xe2x80x94OCH(CO2R5)2,
c) xe2x80x94OCH2(CO2R5),
d) xe2x80x94OCH(CO2R5)CH2CO2R5,
e) xe2x80x94OC(CO2R5)xe2x95x90CHCO2R5,
f) xe2x80x94CH2CH(CO2R5)2,
g) xe2x80x94CHxe2x95x90C(CO2R5)2,
h) xe2x80x94OCH2CONHOH,
i) xe2x80x94N(CH2CO2R5)2, or
j) xe2x80x94OCHF(CO2R5);
wherein R2 is
a) xe2x80x94C1-C10 alkyl optionally substituted with one or two xe2x80x94CO2R5 bonded to the same or different carbon atoms or with one xe2x80x94COxe2x80x94NH2,
b) xe2x80x94C3-C8 cycloalkyl optionally substituted with one xe2x80x94CO2R5,
c) xe2x80x94C0-C6 alkyl-phenyl optionally substituted with one or two xe2x80x94CO2R5 bonded to the same or different carbon atoms or with xe2x80x94CH2CH(CO2R5)2,
d) xe2x80x94CH(R7)NHXR6, or
e) 
wherein R3 is
a) xe2x80x94C1-C12 alkyl, optionally substituted with one to three xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Nxe2x80x94, xe2x80x94Oxe2x80x94C1-C4 alkyl, xe2x80x94Sxe2x80x94C1-C4 alkyl, xe2x80x94Oxe2x80x94G3, xe2x80x94Sxe2x80x94G3, or xe2x80x94OH,
b) xe2x80x94C1-C4 alkyl-C3-C6 cycloalkyl,
c) xe2x80x94C2-C12 alkenyl,
d) xe2x80x94C3-C12 alkynyl,
e) xe2x80x94C0-C10 alkyl(G3)n wherein alkyl is optionally substituted with one to three xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94or xe2x80x94Nxe2x80x94, or
f) xe2x80x94CH(CONH2)C1-C12 alkyl;
wherein R4 is
a) xe2x80x94H,
b) xe2x80x94C1-C18 alkyl or alkenyl, or
c) xe2x80x94C0-C6-alkyl-G3;
wherein R5 is
a) xe2x80x94H,
b) xe2x80x94C1-C10 alkyl, or
c) xe2x80x94C1-C5 alkyl-phenyl;
wherein R6 is
a) C1-C10 alkyl,
b) C0-C6 alkyl-G3,
c) C1-C6 alkyl CONH2,
d) C1-C6 alkyl NHCO2R5,
e) C1-C6 alkyl-OR5,
f) C1-C6 alkyl-NHSO2Me,
g) C1-C6 alkyl-Oxe2x80x94G3,
h) C1-C6 alkyl-Sxe2x80x94G3, or
i) xe2x80x94C1-C6 alkyl-CO2R5;
wherein R7 is
a) xe2x80x94H,
b) xe2x80x94C1-C6 alkyl-G3 
c) xe2x80x94C1-C6 alkyl-CO2R5 
d) C1-C6 alkyl CONH2,
e) C1-C6 alkyl NHCO2R5,
f) C1-C10 alkyl,
g) C1-C10 cycloalkyl,
h) xe2x80x94C1-C6 alkyl-SR5, or
i) xe2x80x94C1-C6 alkyl-S(xe2x95x90O)R5;
wherein R8 is
a) C0-C6 alkyl-G3,
b) CH(R7)CO2R5,
c) CH(R7)CH2CO2R5, or
d) CH(R7)CONHCH2CO2R5;
wherein G3 is
a) phenyl substitued by zero (0) to three (3) R9,
b) naphthyl substitued by zero (0) to three (3) R9, or
c) het substituted by zero (0) to three (3) R9;
wherein het is a 5- or 6-membered saturated or unsaturated ring containing from one (1) to four (4) heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur; and including any bicyclic group in which any of the above heterocyclic rings is fused to a benzene ring, C3-C8 cycloalkyl, or another heterocycle; and if chemically feasible, the nitrogen and sulfur atoms may be in the oxidized forms;
wherein R9 may be any of the following:
a) C1-C8 alkyl substituted by zero (0) to three (3) halo,
b) C2-C8 alkenyl,
c) OH,
d) Oxe2x80x94C1-C5 alkyl,
e) Oxe2x80x94C0-C5 alkyl-phenyl
e) xe2x80x94(CH2)nxe2x80x94Oxe2x80x94C1-C5 alkyl substituted by zero (0) to three (3) hydroxy,
f) xe2x80x94(CH2)nxe2x80x94Oxe2x80x94C2-C7 alkenyl substituted by zero (0) to three (3) hydroxy,
g) halo,
h) NH2,
i) amino-C1-C5 alkyl,
j) mono-or di-C1-C5 alkylamino,
k) xe2x80x94C(O)xe2x80x94C1-C5 alkyl,
l) xe2x80x94CHO,
m) xe2x80x94C(O)xe2x80x94C0-C5 alkyl-phenyl
n) xe2x80x94COOR5 
o) xe2x80x94CON(R5)2,
p) xe2x80x94C3-C7 cycloalkyl,
q) xe2x80x94NO2,
r) xe2x80x94CN,
s) xe2x80x94SO3H,
t) xe2x80x94SO2N(R5)2,
u) xe2x80x94O[(CH2)2xe2x80x94O]nxe2x80x94CH3,
v) xe2x80x94[CH2xe2x80x94O]nxe2x80x94C1-C3 alkyl,
w) xe2x80x94NR5(CO)xe2x80x94NR5,
x) xe2x80x94CF3,
y) xe2x80x94NR5(CO)C1-C5 alkyl,
z) xe2x80x94N(R5)xe2x80x94SO2xe2x80x94R5,
a1) xe2x80x94Oxe2x80x94C(O)xe2x80x94R5,
b1) xe2x80x94S(O)xe2x80x94R5,
c1) xe2x80x94SR5, or
d1) xe2x80x94SO2xe2x80x94R5;
wherein R10 is
a) xe2x80x94H,
b) CO2R5,
c) CONHOH,
d) 5-tetrazolyl,
e) F, or
f) OCH2CO2R5;
wherein R11 is
a) H, or
b) methyl;
wherein X is xe2x80x94COxe2x80x94 or xe2x80x94SO2xe2x80x94 or xe2x80x94CO2xe2x80x94;
wherein n is zero, one, two or three;
or a pharmaceutically acceptable salt thereof;
provided that when R10 is H, R1 is other than xe2x80x94OCH2(CO2R5).
The present invention particularly provides the compounds of formula III or IV 
wherein G1 is
a) xe2x80x94CH(CH2phenyl)NHCO2t-Bu,
b) xe2x80x94CH(CH2phenyl)NHCOC1-C3 alkyl-G3,
c) xe2x80x94CH(CH2phenyl)NHCOC1-C3 alkyl-CO2R5,
d) 
wherein R3 is
a) xe2x80x94C5-C6 alkyl, or
b) xe2x80x94C3-C6 alkyl-phenyl;
wherein R5 is xe2x80x94H;
wherein the configuration of the chiral center(s) is (S).
The compounds of the present invention are named according to the IUPAC or CAS nomenclature system.
The carbon atoms content of various hydrocarbon-containing moieties is indicated by a prefix designating the minimum and maximum number of carbon atoms in the moiety, i.e., the prefix Ci-Cj indicates a moiety of the integer xe2x80x9cixe2x80x9d to the integer xe2x80x9cjxe2x80x9d carbon atoms, inclusive. Thus, for example, C1-C3 alkyl refers to alkyl of one to three carbon atoms, inclusive, or methyl, ethyl, propyl, and isopropyl, straight and branched forms thereof.
Also, the carbon atom content of various hydrocarbon-containing moieties of the present invention may be indicated by a subscripted integer representing the number of carbon and hydrogen atoms in the moiety, e.g., xe2x80x9cCnH2nxe2x80x9d indicates a moiety of the integer xe2x80x9cnxe2x80x9d carbon atoms, inclusive, and the integer xe2x80x9c2nxe2x80x9d hydrogen atoms, inclusive. Thus, for example, xe2x80x9cCnH2nxe2x80x9d wherein n is one to three carbon atoms, inclusive, and two to six hydrogen atoms, inclusive, or methyl, ethyl, propyl and isopropyl, and all isomeric, straight and branched forms thereof.
Examples of alkyl of one to nine carbon atoms, inclusive, are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, and nonyl, and all isomeric forms thereof and straight and branched forms thereof.
Examples of alkenyl of one to five carbon atoms, inclusive, are ethenyl, propenyl, butenyl, pentenyl, all isomeric forms thereof, and straight and branched forms thereof.
By xe2x80x9chaloxe2x80x9d is meant the typical halogen atoms, such as fluorine, chlorine, bromine, and iodine.
The present invention encompasses all possible combinations of configurations at each of the possible chiral centers. The preferred configuration for the chiral center depicted in formula I is (S), and the preferred configuration for the chiral center present in R2 (d and e) is (S).
The compounds of formulae I and II of the present invention are prepared as described in the Charts, Preparations and Examples below, or are prepared by methods analogous thereto, which are readily known and available to one of ordinary skill in the art of organic synthesis.
Commercially available tyrosine benzyl ester A-1 is acylated with monomethyl succinate under standard amide coupling conditions employing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) as the coupling reagent (Tet. Lett. 1993, 34:7685) to afford A-2. Alkylation of the phenol is effected with diethylchloromalonate in acetone with potassium carbonate as catalyst, conditions analogous to those previously described for alkylation of phenols (J. Am. Chem. Soc. 1951, 73:872). Standard hydrogenolysis of the benzyl ester A-3 affords A-4, which is then acylated with various amines (R3NH2) under the influence of EDC. The target acids A-5 are obtained by saponification.
Commercially available Cbz-tyrosine (B-1) is coupled with n-pentylamine under standard EDC conditions, affording amide B-2. Alkylation of the phenol as described in Chart A gives ether B-3, which is then hydrogenolytically deprotected to obtain amine B-4, isolated as the corresponding HCl salt. Acylation of the free amine with various carboxylic acids R2COOH is accomplished with EDC. Final saponification with dilute lithium hydroxide followed by acidification gives the sparingly soluble malonic acids B-5.
Amine hydrochloride B-4 (from Chart B) is acylated with various isocyanates in the presence of triethylamine in methylene chloride to afford the corresponding urethanes. Saponification of the esters then provides the acids C-1.
Amine B-4 (from Chart B) is converted to the corresponding isocyanate D-1 by reaction with diphosgene and Proton Sponge at 0 C. (J. Org. Chem. 1996, 61:3883). Addition of N-benzylglycine ethyl ester followed by saponification then affords the desired urethane triacid D-2.
Commercially available Boc-(L)-tyrosine E-1 is coupled with n-pentylamine, as described for Chart B, to give E-2. The Boc group is removed with HCl in acetic acid, and the resulting amine E-3 is coupled with a mono succinate ester as described for Chart A. The resulting phenol amides E-4 and E-5 are added directly to a dialkyl acetylenedicarboxylate in the presence of triethylamine (Aust. J. Chem. 1995, 48:677). Fumarate ester E-6 is hydrogenated with 10% palladium on carbon to give the saturated triacid E-8. Alternatively, fumarate ester E-7 is saponified to give the unsaturated triacid E-9.
Amine hydrochloride E-3 (from Chart E) is reacted with succinic anhydride in the presence of triethylamine to afford the acid F-1. Sulfation of the phenol is effected with sulfur trioxide/pyridine complex in DMF (Int. J. Pep. Prot. Res. 1990, 35:566) and purification is accomplished with reverse phase HPLC to give F-2.
Previously described E-2 (from Chart E) is treated with trifluoromethanesulfonic anhydride in the presence of pyridine to afford triflate G-1. Palladium-catalyzed cross-coupling of G-1 with tributyl(vinyl)tin affords G-2. G-2 is then ozonized followed by reduction with dimethyl sulfide to give G-3. This aldehyde is then condensed with dibenzylmalonate in the presence of piperidine acetate to afford G-4. Deprotection of the Boc group with saturated HCl/HOAc affords G-5 which is subsequently reacted with succinic anhydride to afford G-6. Hydrogenation of G-6 with H2 and 10% Pd/C gives final triacid G-7.
Direct saponfication of dibenzylester G-6 (from Chart G) affords the unsaturated triacid H-1.
Alkylation of phenol E-4 (from Chart E) is accomplished by a carbenoid insertion reaction with di-t-butyl diazomalonate (Synthesis 1974, 347) catalyzed by rhodium acetate (J. Med. Chem. 1995, 38:4270), affording malonate ether I-1. Removal of the t-butyl esters is accomplished with trifluroacetic acid in methylene chloride, and the benzyl ester is removed by hydrogenolysis, affording the desired triacid I-3.
Amide E-2 (from Chart E) is alkylated on the phenolic hydroxyl with dibenzyl bromomalonate as described for Chart A (potassium carbonate/acetone) to give J-3. The Boc group is removed with HCl in acetic acid, affording the amine hydrochloride J-4. The free amine is added to various cyclic anhydrides in the presence of triethylamine, giving acids J-5. Hydrogenolysis of the benzyl esters then affords the desired triacids J-6.
Chart K describes an alternative synthesis of A-5 (from Chart A) (now K-6 in Chart K) wherein benzyl esters are used as the protecting group for the malonate carboxyls instead of ethyl esters. Tyrosine t-butyl ester K-1 is acylated with monobenzyl succinate under the influence of EDC to afford amide K-2. Alkylation with dibenzyl bromomalonate under the conditions described in Chart A affords ether K-3. The t-butyl ester is removed with TFA in methylene chloride, giving carboxylic acid K-4, which is coupled with various amines using EDC as the coupling reagent. Final deprotection of K-5 is accomplished by hydrogenolysis to give K-6.
Chart L describes an extension of Chart J wherein amine J-4 (from chart J) is coupled (EDC) with a protected amino acid to afford L-2. The Boc group is removed with HCl in acetic acid to give amine L-3. Addition to succinic anhydride followed by hydrogenolysis of the benzyl esters L-4 then provides the desired tetracids L-5.
Cbz-tyrosine M-1 is coupled (EDC) with norleucine amide to provide M-2. Alkylation of the phenol with diethyl chloromalonate as described for Chart A gives ether M-3. The Cbz group is removed by hydrogenation, and the resulting free amine M-4 is acylated with succinic anhydride. Carboxylic acid M-5 is then saponified to give the target triacid M-6.
Commercially available N-1 is condensed with dibenzylmalonate in the presence of piperidine acetate to afford N-2. N-2 is coupled to previously described J-4 (from Chart J) to afford N-3. Hydrogenation of N-3 leads to final tetraacid N-4.
Direct hydrogenation of benzyl ester L-2 (from Chart L) gives the Boc-protected triacid O-1.
Acylation of amine L-3 (from Chart L) with hexanoyl chloride gives amide P-1. Hydrogenation then removes the benzyl esters, providing triacid P-2.
Commercially available Q-1 is N-protected as the Boc derivative by reaction with Boc2O, and the resulting compound is converted to amylamide Q-2 by coupling (EDC) with amylamine. Palladium catalyzed carbonylation with carbon monoxide and methanol affords methyl ester Q-3. Alkylation of the phenolic oxygen with methylbromoacetate yields ether Q-4, which is N-deblocked with trifluoroacetic acid in methylene chloride and then acylated with succinic anhydride, leading to amide Q-5. Saponification under standard conditions then produced the desired triacid Q-6.
Q-4 is deblocked with HCl/dioxane before coupling with 3-phenylpropanoic acid in the presence of EDC and saponified to afford R-4. Alternatively, Q-4 may be deblocked as before, followed by coupling with Boc-L-Phe to afford R-1. R-1 may be saponified directly to R-2, or the Boc group can be removed with HCl/dioxane, and the resulting amine can be coupled with an acid chloride or carboxylic acid to afford R-3 after saponification.
S-1 is alkylated with diethylchloromalonate to afford S-2. Removal of the Boc group with HCl/dioxane followed by coupling with Boc-L-p-benzoyl-Phe gives S-3. Removal of the Boc group again followed by addition of succinic anhydride and saponification provides triacid S-4.
Iodotyrosine Q-2 is converted to nitrile T-1 by the action of zinc cyanide and Pd catalyst. Alkylation with methyl bromoacetate affords ether T-2, which is coupled with Boc-L-Phe after deblocking of the amine group with HCl. The nitrile is converted to the corresponding tetrazole T-4 with TMS-azide and catalytic dibutyltin oxide. Final saponification affords the acid T-5.
Q-2 is carbonylated with carbon monoxide and palladium catalyst to afford esters U-1 and Q-3. The phenols are alkylated with methyl or benzyl bromoacetate to afford U-2 and U-3. The Boc group is removed with TFA, followed by coupling with Boc-L-Phe, affording amides U-4 and U-5. Catalytic hydrogenation removes the benzyl esters, providing U-6 and U-7. Coupling of the free carboxylic acids with hydroxylamine generates the hydroxamic acids U-8 and U-9, and the methyl esters are saponified with lithium hydroxide to provide acids U-10 and U-1.
Ester V-1 is reduced with DIBAL to afford aldehyde V-2, which is subsequently converted by a Wittig reaction to olefin V-3. The phenol is alkylated with dibenzyl bromomalonate to afford ether V-4. Deprotection of the amine with TFA, followed by acylation of the free amine with mono-benzylsuccinate affords amide V-5. Saponification of the esters (LiOH) then provides the triacid V-6.
Commercially available acid W-1 is amidated with n-pentylamine (EDC), and the resulting amide W-2 is catalytically hydrogenated to aniline W-3. The aniline is bis(alkylated) with methyl bromoacetate to afford W-4. Removal of the Boc group (TFA) followed by acylation of the amine with Boc-L-Phe affords W-5. Final saponification (LiOH) then provides the diacid W-6.
Commercially available meta-iodotyrosine is esterified with benzyl alcohol before coupling with Boc-L-Phe, affording X-2. The iodine is carboxylated with CO under palladium catalysis, providing ester X-3, which is alkylated with methyl bromoacetate. The resulting ether X-4 is hydrogenated to remove the benzyl ester protecting group, and the resulting acid X-5 is reduced with sodium borohydride via the corresponding acyl imidazole to alcohol X-6. Final saponification of the esters affords the diacid X-7.
Commercially available methyl tyrosine Y-1 is protected as the N-Boc derivative under standard conditions before conversion of the carboxylic acid to amide Y-3. This amide is alkylated with dibenzyl bromomalonate to afford ether Y-4. Boc cleavage with HCl is followed by acylation of the free amine with succinic anhydride, providing acid Y-5. Final saponification with LiOH affords triacid Y-6.
Meta fluorotyrosine Z-1 is converted to triacid Z-6 exactly as described in Chart Y.
4-Hydroxybenzaldehyde AA-1 is alkylated with diethyl chloromalonate to afford ether AA-2. Mono ethyl malonate AA-3 is coupled with amylamine under standard conditions (DEPC) to afford amide AA-4. Hydrolysis of the ester with aq NaOH provides acid AA-5. Coupling of AA-5 with beta-alanine ethyl ester provides malondiamide AA-6, which is condensed with aldehyde AA-2 under Knoevenagel conditions. The resulting methylidene malondiamide AA-7 (a mixture of olefin isomers) is saturated by catalytic hydrogenation, and the ester AA-8 is saponified to triacid AA-9 with aq NaOH.
Amine B-4 (from Chart B) is acylated with the appropriate protected amino acid under the influence of EDC and triethylamine. The resulting amides BB-1 is directly saponified to give BB-3. Alternatively, where R6 is t-butylcarboxy (Boc), the Boc group is removed with HCl/acetic acid, and the resulting free amine acylated with succinic anhydride. Final saponification then affords the triacids BB-2.
Diol CC-1 (reference given in Example 142) is bis(alkylated) with ethyl bromoacetate, and the resulting bis(ether) CC-2 is hydrogenated to remove the benzyl ester. The carboxylic acid CC-3 is coupled with amylamine under standard conditions (DEPC) before cleavage of the Boc group with TFA. Free amine CC-5 is then coupled with Boc-L-Phe (DEPC), and the resulting amide CC-6 is saponfied to the diacid CC-7.
Diester Q-4 (Chart Q) is reacted with TFA to cleave the Boc group, and the free amine DD-1 is coupled (DEPC) with the appropriate amino acid (see Example 143) to afford amide DD-2. Saponification provides diacid DD-3.
Q-2 (Chart Q) is carbonylated with CO under palladium catalysis to afford ester EE-1. The phenol is alkylated with ethyl bromofluoroacetate/potassium carbonate to afford ether EE-2. Boc deprotection and amide coupling of the free amine with Boc-L-Phe under standard conditions affords diester EE-3, which is saponified to provide the diacid EE-4.
Acid X-5 (Chart X) is coupled with 4-phenylbutylamine under standard amide coupling conditions to provide FF-1. Saponification of the esters affords diacid FF-2.
Preferred methods of preparation are depicted in Charts A, B, BB, Q and R.
The present invention provides for compounds of formulae I and II or pharmacologically acceptable salts and/or hydrates thereof. Pharmacologically acceptable salts refers to those salts which would be readily apparent to a manufacturing pharmaceutical chemist to be equivalent to the parent compound in properties such as formulation, stability, patient acceptance and bioavailability. Examples of salts of the compounds of formula I include lithium, sodium and potassium.
Where R5 is other than H, the compounds would not be expected to have intrinsic activity, but would be expected to possess activity in vivo following hydrolysis by non-specific esterases to the corresponding carboxylic acids.
The compounds of the present invention are useful for treating patients with noninsulin-dependent diabetes mellitus (NIDDM) and related diseases. For this indication, these compounds may be administered by oral, intranasal, transdermal, subcutaneous and parenteral (including intramuscular and intravenous) routes in doses of 0.1 mg to 1000 mg/kg of body weight per day.
Those skilled in the art would know how to formulate the compounds of this invention into appropriate pharmaceutical dosage forms. Examples of the dosage forms include oral formulations, such as tablets or capsules, or parenteral formulations, such as sterile solutions.
When the compounds in this invention are administered orally, an effective amount is from about 0.1 mg to 100 mg per kg of body weight per day. Either solid or fluid dosage forms can be prepared for oral administration. Solid compositions, such as compressed tablets, are prepared by mixing the compounds of this invention with conventional ingredients such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, acacia, methyl cellulose, or functionally similar pharmaceutical diluents and carriers. Capsules are prepared by mixing the compounds of this invention with an inert pharmaceutical diluent and placing the mixture into an appropriately sized hard gelatin capsule. Soft gelatin capsules are prepared by machine encapsulation of a slurry of the compounds of this invention with an acceptable inert oil such as vegetable oil or light liquid petrolatum.
Syrups are prepared by dissolving the compounds of this invention in an aqueous vehicle and adding sugar, aromatic flavoring agents and preservatives. Elixirs are prepared using a hydroalcoholic vehicle such as ethanol, suitable sweeteners such as sugar or saccharin and an aromatic flavoring agent. Suspensions are prepared with an aqueous vehicle and a suspending agent such as acacia, tragacanth, or methyl cellulose.
When the compounds of this invention are administered parenterally, they can be given by injection or by intravenous infusion. An effective amount is from about 0.1 mg to 100 mg per kg of body weight per day. Parenteral solutions are prepared by dissolving the compounds of this invention in aqueous vehicle and filter sterilizing the solution before placing in a suitable sealable vial or ampule. Parenteral suspensions are prepared in substantially the same way except a sterile suspension vehicle is used and the compounds of this invention are sterilized with ethylene oxide or suitable gas before it is suspended in the vehicle.
The exact route of administration, dose, or frequency of administration would be readily determined by those skilled in the art and is dependant on the age, weight, general physical condition, or other clinical symptoms specific to the patient to be treated.
The utility of representative compounds of the present invention has been demonstrated in the biological assays described below:
PTP1 Assays: A construct, which consisted of a C-terminal truncation of rat PTP1 (amino acid residues 1-322) (cloned from a rat brain library) with an N-terminal glutathione S-transferase (GST) tag and an adjacent thrombin cleavage site, was inserted into vector plasmid pGEX-2T and transformed into E.coli strain TG-1 under the control of a lac promoter (K. L. Guan and J. E. Dixon (1991) Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Analyt. Biochem. 192: 262-267). The GST-fusion protein was purified on a glutathione agarose affinity column, the GST tag was cleaved with thrombin, and the active enzyme was recovered for use in an assay to identify PTP inhibitors.
The equivalent construct of human PTP1B (amino acid residues 1-321) (cloned from a human placental library), without the GST tag and thrombin cleavage site, was inserted into a pMB replicon and transformed into E. coli BL21(DE3), a strain containing a chromosomal copy of the gene for T7 RNA polymerase under control of a lacUV5 promoter. Expression of PTP1B was induced with isopropyl thiogalactose and the soluble protein was purified by ion exchange, hydrophobic interaction and gel exclusion chromatography for use in the assay to identify PTP inhibitors.
PTP1 activity is measured using either p-nitrophenol phosphate (pNPP) or a triphosphopeptide (that matches residues 1142 through 1153 of the xcex2-subunit and the insulin receptor) as substrate in a 96-well microtiter plate format. An assay pH of 7.2 is used for standard assays (measured 405=9800 at pH 7.2).
Human PTP1B, which is highly homologous to rat PTP1, was assayed exactly as described above for PTP1. The PTP1 inhibitors described here also inhibit PTP1B with similar or identical potencies.
Standard assays are conducted at room temperature in a total volume of 0.2 ml that contains Hepes buffer (50 mM, pH 7.2), NaCl (50 mM), EDTA (1 mM), DTT (1 mM), bovine serum albumin (1 mg/ml), pNPP (1 mM) and PTP1 (35 ng/ml). Compounds (2 xcexcl of 10 mM solutions) are pipetted into wells of microtiter plates followed by 198 xcexcl of premixed reaction mix (with PTP1 and pNPP added immediately before use). The rate of change in A405 is recorded for 60 min. Two wells on each plate contain DMSO controls and two wells contain sodium orthovanadate (1 mM) which inhibits PTP1-catalyzed hydrolysis of pNPP completely. Data are expressed as percent inhibition relative to the average of the DMSO controls measured on the same microtiter plate.
When triphosphopeptide1142-1153 is used as substrate, the rate of release of inorganic phosphate is measured using a Malachite Green/phosphomolybdate reaction (A. A. Baykov, O. A. Evtushenko, and S. M. Avaeva (1988) A Malachite Green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay. Anal. Biochem. 171: 266-270.) in a microtiter plate format. Standard assays are conducted at room temperature in a total volume of 50 xcexcl that contains Hepes buffer (50 mM, pH 7.2), NaCl (50 mM), EDTA (1 mM), DTT (1 mM), bovine serum albumin (1 mg/ml), triphosphopeptide1142-1153 (200 xcexcM) and PTP1 (87 ng/ml). Reactions are terminated with the addition of 0.15 ml of Malachite Green/ammonium molybdate reagent [10 ml Malachite Green (0.44 g in 6N H2SO4), 2.5 ml ammonium molybdate (7.5% w/v), 0.2 ml Tween 20 (11% w/v)] that is diluted with 8 parts of water immediately before use, and after 1 h absorbance at 650 nm is measured. The phosphate assay is calibrated using either KH2PO4 or pNPP (after ashing with Mg(NO3)2) which gives essentially identical standard curves. The phosphate assay is useful in the range of 1 to 10 nmol Pi.
The % inhibition of pNPP-hydrolysis by compounds of the present invention are listed in Table 1 and 2 below.
The following compounds of the present invention are preferred:
(S)-5-[[[[1-[[4-(Dicarboxymethoxy)phenyl]methyl]-2-oxo-2-(pentylamino)ethyl]amino]carbonyl]amino]-1,3-benzenedicarboxylic acid;
(S)xe2x80x94N-[[[1-[[4-(Dicarboxymethoxy)phenyl]methyl]-2-oxo-2-(pentylamino)ethyl]amino]carbonyl]-L-glutamic acid;
N-[(1,1-Dimethylethoxy)carbonyl]-L-xcex1-aspartyl-O-(dicarboxymethyl)-N-pentyl-L-tyrosinamide;
N-(3-Carboxy-1-oxopropyl)-L-xcex1-aspartyl-O-(dicarboxymethyl)-N-pentyl-L-tyrosinamide;
N-(3-Carboxy-1-oxopropyl)-L-xcex1-glutamyl-O-(dicarboxymethyl)-N-pentyl-L-tyrosinamide;
N-(3-Carboxy-1-oxopropyl)-O-(dicarboxymethyl)-L-tyrosyl-L-norleuciramide;
N-(1-Oxohexyl)-L-xcex1-aspartyl-O-(dicarboxymethyl)-N-pentyl-L-tyrosinamide;
N-[(Phenylmethoxy)carbonyl]-L-xcex1-aspartyl-O-(dicarboxymethyl)-N-pentyl-L-tyrosinamide;
N-[(1,1-Dimethylethoxy)carbonyl]-D-xcex1-aspartyl-O-(dicarboxymethyl)-N-pentyl-L-tyrosinamide;
4-Benzoyl-N-(3-carboxy-1-oxopropy)-L-phenylalanyl-O-(dicarboxymethyl)-N-pentyl-L-tyrosinamide; and
(S)-2-(Carboxymethoxy)-5-[2-[(3-carboxy-1-oxopropyl)amino]-3-oxo-3-(pentylamino)propyl]benzoic acid.
The following compounds of the present invention are more preferred:
2-{4-[(2S)-2-({(2S)-2-[(3-carboxypropanoyl)amino]-3-phenylpropanoyl}amino)-3-oxo-3-(pentylamino)propyl]phenoxy}malonic acid;
5-[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-phenylpropanoyl}amino)-3-oxo-3-(pentylamino)propyl]-2-(carboxymethoxy)benzoic acid;
2-{4-[(2S)-2-{[(dibenzylamino)carbonyl]amino}-3-oxo-3-(pentylamino)propyl]phenoxy}malonic acid;
2-(carboxymethoxy)-5-[(2S)-2-{[(dibenzylamino)carbonyl]amino}-3-oxo-3-(pentylamino)propyl]benzoic acid;
2-(carboxymethoxy)-5-[(2S)-2-({(2S)-2-[(3-carboxypropanoyl)amino]-3-phenylpropanoyl}amino)-3-oxo-3-(pentylamino)propyl]benzoic acid;
2-(carboxymethoxy)-5-{(2S)-3-oxo-3-(pentylamino)-2-[((2S)-3-phenyl-2-{[2-(5-sulfanyl-1H-1,2,3,4-tetraazol-1-yl)acetyl]amino}propanoyl)amino]propyl }benzoic acid;
2-(carboxymethoxy)-5-{(2S)-3-oxo-3-(pentylamino)-2-[((2S)-3-phenyl-2-{[2-(1H-1,2,3-triazol-5-ylsulfanyl)acetyl]amino}propanoyl)amino]propyl }benzoic acid;
2-[4-[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-phenylpropanoyl}amino)-3-oxo-3-(pentylamino)propyl]-2-(2H-1,2,3,4-tetraazol-5-yl)phenoxy]acetic acid;
2-(carboxymethoxy)-5-[(2S)-3-oxo-3-(pentylamino)-2-({(2S)-3-phenyl-2-[(2-phenylacetyl)amino]propanoyl}amino)propyl]benzoic acid;
2-(carboxymethoxy)-5-[(2S)-3-oxo-3-(pentylamino)-2-({(2S)-3-phenyl-2-[(4-phenylbutanoyl)amino]propanoyl}amino)propyl]benzoic acid;
2-(carboxymethoxy)-5-[(2S)-2-({(2S)-2-[(3-methoxypropanoyl)amino]-3-phenylpropanoyl}amino)-3-oxo-3-(pentylamino)propyl]benzoic acid;
2-(carboxymethoxy)-5-((2S)-3-oxo-3-(pentylamino)-2-{[(2S)-3-phenyl-2-({2-[4-(trifluoromethyl)phenyl]acetyl}amino)propanoyl]amino}propyl)benzoic acid;
2-(carboxymethoxy)-5-[(2S)-2-[((2S)-2-{[2-(4-methoxyphenyl)acetyl]amino}-3-phenylpropanoyl)amino]-3-oxo-3-(pentylamino)propyl]benzoic acid;
2-(carboxymethoxy)-5-[(2S)-3-oxo-3-(pentylamino)-2-({(2S)-3-phenyl-2-[(3-phenylpropanoyl)amino]propanoyl}amino)propyl]benzoic acid;
2-(carboxymethoxy)-5-[(2S)-3-oxo-2-{[(2R)-2-(2-oxo-1-pyrrolidinyl)-3-phenylpropanoyl]amino}-3-(pentylamino)propyl]benzoic acid; and
5-{(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-phenylpropanoyl}amino)-3-[(4-phenylbutyl)amino]propyl}-2-(carboxymethoxy)benzoic acid.